DNA damage caused by alkylating agents is recognized as an initiator of cell killing and carcinogenesis. Most cells have a similar potential to avoid these events through the coordinated action of proteins that repair DNA, yet various mammalian cell types show marked differences in viability after exposure to an alkylating agent. In the model organism Saccharomyces cerevisiae the candidate has shown that large protein networks anchored to repair pathways influence cellular viability after challenge from an alkylating agent. This suggests that accessory proteins found in a cell have prominent roles in coordinating DNA repair in vivo. Therefore, the hypothesis that human proteins that interact with key DNA repair pathways are critical for preventing alkylation induced cytotoxicity will be tested. To generate a cell specific map of protein interaction networks that influence DNA repair and gain a better understanding of factors that influence carcinogenesis and cell death, the following three aims will be pursued. First, the phenotvpe of cells that have RNA interference (RNAi) based knock-downs of important DNA repair proteins will be determined, after treatment with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). This will systematically characterize the alkylation phenotype of one human cell type containing specific repair-protein deficiencies. Secondly, the pre-and post-treatment (MMS and MNU) protein-protein interactions for this group of important DNA repair proteins will be identified. Experiments will be performed using yeast two-hybrid screens and mass spectrometry based identification of protein complexes to identify networks originating from DNA repair pathways. Importantly, this will allow the identity of basal and induced protein-protein interactions. Resultant data will generate a protein map of the dynamic interactions associated with cellular responses to mutagens. Lastly, alkylation resistance networks using RNAi based phenotyping of interacting partners and DNA repair assays will be constructed. Data will be collected in a database, computationally compiled, and used to guide experiments that systematically test knock-downs for an alkylation phenotype and DNA repair activity. Collectively, results will allow: 1) to test the hypotheses, 2) generate a cell specific map of protein interaction networks in DNA repair, and 3) systematically explore the human genome for accessory proteins important for DNA repair.